Facsdiva version 6

Mesenchymal stem cells were detached from culture dish using TrypLE™ express (Thermo Fisher Scientific, Waltham, MA) and collected in Eppendorf tubes. The cells were stained for MSC markers using anti-CD44, PE-Cy5 (BD Biosciences, Franklin Lakes, NJ, #553135), anti-CD73, BV605 (Biolegend, San Diego, CA, #127215), anti-CD105, AF647 (Biolegend, San Diego, CA, #120405), and anti-E-selectin/CD62E, PE (BD Biosciences, Franklin Lakes, NJ, #553751) to assess E-selectin levels. The cells were analyzed on a on a FACSAria II cell sorter using FACSDiva Version 6.1.1 (BD Biosciences) or FlowJo Version 7.6.4 (TreeStar) software.

FACS analysis was carried out on a FACSaria II cell sorter (BD, Franklin Lakes, NJ, USA). Appropriate lasers and filters were used for all fluorochromes included. Data analysis was conducted using FACSdiva version 6.1.1 (BD). An overview of the gating strategy is provided in S2 Fig. Background antibody staining was determined by appropriate isotypic controls. For SP and Main Population (MP), gating was conducted using the most effective inhibitor (verapamil, and sometimes sodium azide/2-Deoxy-D-glucose) to set a morphological SP and MP gate, respectively. Isotypic and inhibitor controls were subtracted when statistics were calculated. After sorting, cell samples were stored in a freezer at –80° C until further analysis.

Fluorescence-activated cytometry was performed as described [27] (link), [38] (link). Quantum Simply Cellular calibration beads that contain four Quantum Simply Cellular microsphere populations with different mouse IgG antibody binding capacities were stained with fluorophore-conjugated monoclonal antibodies specific for IL12Rβ1, IL12Rβ2, or HLA-ABC. The cells were analyzed using a FACSAria flow cytometer and FACSDiva Version 6.1.1 software (BD Biosciences). No stain controls were used as negative flow cytometry controls. Single stain controls were used to establish fluorescent compensation parameters. Cellular events were identified by forward and side scatter characteristics. On average, events were analyzed. Flow cytometry data was exported as FCS3.0 files and analyzed using R/Bioconductor [73] (link).

To test levels of ROS and mitochondrial potential, fluorescent indicator DHE (Invitrogen), DCF (Sigma) and JC-1 (Invitrogen) were used. Cells were harvested, washed in PBS and stained with 5 μM DHE or 2 μM JC-1 dyes for 30 min at 37 °C in the dark. For DCF detection, cells were stained with the dye for 30 min at 37 °C and washed out at 1 h prior to analysis. Fluorescence was measured immediately with LSRFortessa (BD Biosciences), and the data were analyzed with the FACSDiva version 6.2 software (BD Biosciences). The fluorescence intensity of the red signal in DHE-stained cells and green signal in DCF-stained cells indicate the levels of superoxide and hydrogen oxide, respectively. The ratio of the red to green fluorescence intensity in JC-1-stained cells was used to represent mitochondrial membrane potential.

For the analyses of hematopoietic cells, a hypotonic lysis was performed to remove erythrocytes. 50 μl of blood was lysed using 500 μl of ACK (Ammonium-Chloride-Potassium) Lysing Buffer (Lonza, Walkersville) 10 min at room temperature. After the erythrocytes lysis, two washes with PBS were performed prior the incubation with the antibodies for 30 min at 4°C. Cells were stained with the following fluorochrome-coupled antibodies: Allophycocyanin (APC)-conjugated anti-mouse CD11b (1:300; cn.17–0112 eBioscience, USA) to label myeloid cells, phycoerythrin (PE)-conjugated anti-mouse B220 (1:100; cn.12–0452, eBioscience, USA) for B lymphocytes and phycoerythrin/cyanine (PE/Cy7)-conjugated anti-mouse CD3, 1:100; cn.100320, BioLegend, USA) for T lymphocytes. Immunofluorescence of labeled cells was measured using a BD LSR II flow cytometer. Dead cells and debris were excluded by measurements of forward- versus side-scattered light and DAPI (4′,6-diamino-2-phenylindole) (Sigma) staining. Gates for the respective antibodies used were established with isotype controls and positive cell subset controls. Data analysis was carried out using FACSDiva version 6.2 software (BD biosciences).